Mammalian cell membranes are important to the structural integrity and activity of many cells and tissues. Of particular interest in membrane physiology is the study of trans-membrane ion channels which act to directly control a variety of pharmacological. physiological, and cellular processes. Numerous ion channels have been identified including calcium, sodium, and potassium channels, each of which have been investigated to determine their roles in vertebrate and insect cells.
Because of their involvement in maintaining normal cellular homeostasis, much attention has been given to potassium channels. A number of these potassium channels open in response to changes in the cell membrane potential. Many voltage-gated potassium channels have been identified and characterized by their electrophysiological and pharmacological properties. Potassium currents are more diverse than sodium or calcium currents and are further involved in determining the response of a cell to external stimuli.
The diversity of potassium channels and their important physiological role highlights their potential as targets for developing therapeutic agents or various diseases. One of the best characterized classes of potassium channels are the voltage-gated potassium channels. The prototypical member of this class is the protein encoded by the Shaker gene in Drosophila melanogaster. Proteins of the Shal or Kv4 family are a type of voltage-gated potassium channels that underlies many of the native A type currents that have been recorded from different primary cells. Kv4 channels have a major role in the repolarization of cardiac action potentials. In neurons. Kv4 channels and the A currents they may comprise play an important role in modulation of firing rate, action potential initiation and in controlling dendritic responses to synaptic inputs.
The Kv family of channels includes, among others: (1) the delayed-rectifier potassium channels, which repolarize the membrane after each action potential to prepare the cell to fire again; and (2) the rapidly inactivating (A-type) potassium channels, which are active predominantly at subthreshold voltages and and act to reduce the rate at which excitable cells reach firing threshold. In addition to being critical for action potential conduction. Kv channels also control the response for depolarizing, e.g., synaptic, inputs and play a role in neurotransmitter release. As a result of these activities, voltage-gated potassium channels are key regulators of neuronal excitability (Hille B., Ionic Channels of Excitable Membranes. Second Edition, Sunderland. MA; Sinauer. (1992)).
There is tremendous structural and functional diversity within the Kv potassium channel superfamily. This diversity is generated both by the existence of multiple genes and by alternative splicing of RNA transcripts produced from the same gene. Nonetheless, the amino acid sequences of the known Kv potassium channels show high similarity. All appear to be comprised of four, pore forming α-subunits and some are known to have four cytoplasmic (β-subunit) polypeptides (Jan L. Y. et al. (1990) Trends Neurosci 13:415–419, and Pongs, O. et al. (1995) Sem Neurosci. 7:137–146). The known Kv channel α-subunits fall into four sub-families named for their homology to channels first isolated from Drosophila; the Kv1, or Shaker-related subfamily; the Kv2, or Shaw-related subfamily; the Kv3, or Shaw-related subfamily; and the Kv4, or Shal-related subfamily.
Kv4.2 and Kv4.3 are examples of Kv channel α-subunits of the Shal-related subfamily. Kv4.3 has a unique neuroanatomical distribution in that its mRNA is highly expressed in brainstem monoaminergic and forebrain cholinergic neurons, where it is involved in the release of the neurotransmitters dopamine, norepinephrine, serotonin, and acetylcholine. This channel is also highly expressed in cortical pyramidal cells and in interneurons. (Serdio P. et al. (1996)J. Neurophys 75:2174–2179). Interestingly, the Kv4.3 polypeptide is highly expressed in neurons which express the corresponding mRNA. The Kv4.3 polypeptide is expressed in the somatodendritic membranes of these cells, where it is thought to contribute to the rapidly inactivating K+ conductance. Kv4.2 mRNA is widely expressed in brain, and the corresponding polypeptide also appears to be concentrated in somatodendritic membranes where it also contributes to the rapidly inactivating K+ conductance (Sheng et al. (1992) Neuron 9:271–84). These somatodendrite A-type Kv channels, like Kv4.2 and Kv4.3 are likely involved in processes which underlie learning and memory, such as integration of sub-threshold synaptic responses and the conductance of hack-propagating action potentials (Hoffman D. A. et al. (1997) Nature 387:869–875).
Thus, proteins which interact with and modulate the activity of potassium channel proteins e.g., potassium channels having a Kv4.2 or Kv4.3 subunit, provide novel molecular targets to modulate neuronal excitability, e.g., action potential conduction, somatodendrite excitability and neurotransmitter release, in cells expressing these channels. In addition, detection of genetic lesions in the gene encoding these proteins could be used to diagnose and treat cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina.
A.Summary of the Invention-3-B.Brief Description of the Drawings-4-C.Detailed Description of the Invention-8-I.Screening Assays-17-II.Predictive Medicine-24-1.Diagnostic Assays-25-2.Prognostic Assays-26-3.Monitoring of Effects During Clinical Trials-31-III.Methods of Treatment-32-1.Prophylactic Methods-33-2.Therapeutic Methods-33-3.Pharmacogenomics-40-D.Examples-42-